Many Gram bacteria displaying positive Gram staining are subjects of study as a biological model (for example bacteria of the genus Bacillus), as a fermentation strain of industrial importance (lactic acid bacteria) or as a pathogen (for example Clostridia, Listeria, Staphylococcus, Streptococcus). Many of these strains are characterized from a physiological standpoint, but few have been studied or modified genetically. The study or modification of the strains may be facilitated by the use of vectors permitting directed or non-specific insertions into the bacterial chromosome. Delivery systems which are based on the non-replicative vectors are limited to bacteria which can be transformed with a high frequency, and those utilizing replicons which are active only under certain conditions are often limited to their host range. Thus, the construction of recombinant strains requires considerable effort, and can be applied efficaciously only to certain specific microorganisms.
The addition, loss or modification of genes can transform the role of an organism in an industrial process such as fermentation.
Biotechnology seeks to facilitate the industrial use of microorganisms. For example, lactic bacteria are used in agri-foodstuffs, predominantly for the manufacture of fermented dairy products, but also outside the milk industry for the manufacture of wine, cider, cooked meats and silage.
It is hence especially desirable to have effective means available for introducing or modifying specifically and permanently certain genes in these organisms.
At the present time, modification of the chromosome in lactic bacteria is performed via a system by transformation of a non-replicative plasmid. In a single step, it is necessary to have two low-frequency events, transformation with a plasmid and recombination in the chromosome. The probability of obtaining these two events in a single step is the product of the probabilities of each; there is hence a very small chance of obtaining the modification.
Plasmid pWV01 is a cryptic plasmid initially isolated in Lactococcus lactis subsp. cremoris; it is a broad-host-range plasmid which is replicative in both Gram-positive and Gram-negative bacteria, in particular in E. coli, Bacillus subtilis, Lactococcus lactis, Streptococcus and Lactobacillus. It has been characterized, and its nucleotide sequence has been published by Leenhouts et al. (1991).
In Application WO 85/03495, large fragments of this plasmid are used to construct a recombinant plasmid pGK12 marked with the gene for resistance to erythromycin and/or the gene for resistance to chloramphenicol (chloramphenicol acetyltransferase (CAT)). This plasmid pGK12 cannot be used to make integrations in the bacterial chromosome.
The non-replicative plasmids used hitherto enable this problem to be alleviated, but this system requires high degrees of transformation to permit the detection of low-frequency events such as transposition or recombination in the chromosome; now, most lactic bacteria are weakly transformable.
It would be possible to overcome all these difficulties by obtaining a temperature-sensitive replicon which could be used as a delivery vector in lactic or other bacteria.
Plasmids pE194 and pSH71 have been described as naturally temperature-sensitive, above a temperature of 51.degree. C. (J. Bacteriol., 1990, 172, 4543-4548).